Method for detecting, screening, and/or monitoring cancer in an individual

ABSTRACT

The invention relates to a method for screening and/or detecting and/or monitoring a cancer in an individual, said method comprising determining a first parameter represented by the concentration of TIMP-1 in at least one excreta, e.g. saliva, from the individual. The invention provides a method that without the need to use a blood sample is suitable for facilitating the early diagnosis of a cancer, monitoring the recurrence of a cancer, and/or monitoring the status of a cancer or the effect of cancer treatment in an individual.

The present invention relates to a method for early detection and/orscreening and/or monitoring of a cancer in an individual.

A significant factor affecting the long term survival of cancer patientsis the stage at which the cancer is detected. Early detection mayfacilitate rapid and complete removal of any malignancy beforemetastasis occurs as documented by increased cure rate and long-termsurvival of such patients.

Furthermore, if a cancer is detected, an early detection of the spreadof cancer, the recurrence of cancer after treatment as well as theresponse to treatment, is crucial to the survival of cancer patients.

The best method of early detection is routine screening of thoseconsidered to be at risk. However, existing screening methods have metwith variable success. It may be that they are too expensive and/orunreliable to be widely used. In any case, for many cancers, there is nopractical detection method available. This problem, in combination withthe fact that certain cancers, e.g. gastric and colorectal cancers,exhibit symptoms consistent with much more common non-malignantpathologies, make them difficult to detect at an early stage.

Colorectal cancer (CRC) is currently the fourth most common cancer inthe US and ranks second as a cause of cancer-related deaths. Evidenceexists that reductions in colorectal cancer morbidity and mortality canbe achieved through detection and treatment of early-stage colorectalcancers and the identification and removal of adenomatous polyps, theprecursors of colorectal cancer.

Currently, the only way of diagnosing colorectal cancer is to carry outa colonoscopy, which is impractical as a screening technique for generalapplicability. Furthermore, since the disease is still primarily managedby surgical resection, there has been great interest in screening.

A known screening test for colorectal cancer that is often recommendedon a routine basis by many health-care organizations is the fecal occultblood test (FOBT), which uses various methods to detect blood in thestool. However, although the test has a high specificity, this methodhas an intermediate sensitivity often leading to either false-positiveor false-negative results, and may therefore cause unwarranted anxietyabout cancer and unnecessary further tests or may miss the disease inits early stages.

The American Society of Clinical Oncology (ASCO) has thereforerecommended against the use of fecal occult blood tests as a means forregular postoperative monitoring of colorectal cancer, (Benson et al,JCO, 2000). ASCO has also emphasised that new surveillance methods forthe detection of colorectal cancer recurrences are needed (Benson et al,JCO, 2000).

Because metastatic disease is the main cause of cancer patient morbidityand mortality, molecules involved in the regulation of tumour invasionand metastasis are attractive as potentialscreening/detection/monitoring targets. It is well established thatproteolytic enzymes produced by cancer cells or by cells in the tumourstroma are involved in extracellular tissue degradation, leading tocancer cell invasion and metastasis. A number of enzymes have beenassociated with this process, the most thoroughly investigated being themetalloproteinases, such as the collagenases and stromelysins, and theserine proteases such as plasmin. Recently, data has been publishedindicating that these molecules, free or bound in complexes, arereleased from tumour tissue and find their way into the circulation.

A positive correlation between tumour cell aggressiveness and matrixmetalloproteinases (MMPs) expression has been well documented. MMPs area group of zinc-binding endopeptidases involved in connective tissuematrix remodelling and degradation of the extracellular matrix (ECM),which are essential steps in tumour invasion, angiogenesis, andmetastasis (reviewed by Aznavoorian et al, 1993).

However, matrix metalloproteinase enzyme activity is highly regulated.Regulation occurs at multiple levels, including modulation of MMP geneexpression, extracellular activation of the pro-enzyme, inhibition ofactivation and enzymes activity by association with tissue inhibitors ofmetalloproteinase (TIMPS) (Matrisian, 1990), (Mauviel, 1993),(Birkedal-Hansen, 1993).

TIMPs forms stoichiometric complexes with both latent and active MMP's(Welgus et al., 1985; Kleiner et al., 1993), thereby inhibiting thecatalytic activity of these enzymes (Stetler-Stevenson et al., 1996;Goldberg et al., 1992; Birkedal-Hansen et al., 1993).

TIMPs represent a family of ubiquitous proteins. There have, at present,been identified four members of the TIMP family. It has been found thatTIMP-1 and TIMP-2 are capable of inhibiting tumour growth, invasion, andmetastasis (Liotta, et al., 1991 and Khokha et al., 1989). However,recent clinical studies have stated TIMP-1 and TIMP-2 to be rathertumour-promoting molecules, as they were found to be significantlyoverexpressed in patients with poor prognosis. (McCarthy et al, 1999;Remacle et al, 2000).

Studies have shown that TIMP-1 in plasma is a highly sensitive andspecific screening marker for the identification of patients with a highrisk of having colorectal cancer (Holten-Andersen et al., 1999 andHolten-Andersen et al. 2002).

A method using this information is disclosed in WO00/62070, the completedisclosure of which is incorporated herein by reference for allpurposes. In said reference it has been found that individuals can bescreened for colorectal cancer by measuring the TIMP-1 concentration inplasma samples.

However, the method has the drawback that a blood sample must be drawnfor each individual. This can be extremely expensive and troublesomewhen detecting and monitoring cancers in large populations and in somecases even impossible e.g. in the developing countries.

Furthermore, trained staff must draw blood samples, and particular caremust be taken with the samples, as there is a high prevalence ofhepatitis B and the human immunodeficiency virus (HIV) infections insome populations, e.g. in the developing countries.

Based on these problems other fluids mentioned in the exhausted list ofWO/0062070 such as urine and serum has been evaluated as an alternativeto plasma, as these are the traditional biological samples for thequalitative and quantitative measurement of substances includingproteins.

However, serum studies have shown that there is no significantdifference between the total TIMP-1 concentrations in serum samples fromindividuals having colorectal cancer and healthy blood donors, mostproperly because the platelets will leak TIMP-1 during coagulation invitro (Cooper et al, 1985; Jung et al, 1996). Furthermore, in otherstudies, TIMP-1 has by itself been proven inadequate as a serum markerfor screening for pancreatic cancer, (Zhou et al, 1998).

The concentration of TIMP-1 in urine and plasma from the same colorectalcancer patients has also been evaluated, but without finding anynoticeable correlation, since no TIMP-1 elevation was found in the urine(Brünner, unpublished data).

It is known for the person skilled in the art, that all organiccompounds of plasma, such as hormones, immunoglobulines, enzymes,proteins, DNA and viruses may be detected in saliva in trace amounts(Vining and McGinley, 1985). However, it is a well established knowledgethat the concentration found in saliva is lower than the concentrationin urine which again is lower than the concentration found in plasma.

The lower concentrations in saliva are caused by a number of factors. Asan example can be mentioned that saliva contains enzymes for digestionand that the total protein concentration in saliva is negligible sinceit is less than 1% of that in plasma (Breimer and Danhof, 1980).Furthermore it is likely that a major source of these trace amountsoriginates from the gingival crevicular fluid (from the tooth/gummargin) (Cimasoni, 1974).

A person skilled in the art would therefore expect that the naturalenzymes, such as proteases in the saliva, would digest these negligibleprotein concentrations and reduce any concentration found in e.g. salivato a negligible concentration if detectable at all.

One of a number of examples of a protein having a lower concentration inurine than in serum/plasma and again an even lower concentration insaliva, is prostate-specific antigen (PSA), which is the most commonlyused serum marker for screening for prostate cancer, (Balk et al, 2003).PSA is present in high concentrations in serum and plasma, in minorconcentrations in urine, and in one study no detectable PSA levels havebeen found in saliva at all, (Lovgren et al, 1999). Furthermore, anotherstudy has shown that determination of free and total PSA in saliva toimprove and simplify the differentiation between prostate cancer andbenign prostatic hyperplasia is not suitable for use as an alternativemeasurement of serum (Turan et al, 2000).

Accordingly, since no elevated levels of TIMP-1 could be found in urineof either healthy blood donors or individuals suffering from colorectalcancer, the person skilled in the art would based on the common generalknowledge that the concentration of a protein in saliva always would belower than in urine and plasma have no hint to examine saliva as apossible body fluid for detection of TIMP-1.

It should in this respect be noted, that TIMP-1 can be found in sputumfrom patients with asthma and chronic bronchitis (Vigonola et al, 1998),but TIMP-1 is in such cases coming from blood from small lesions in e.g.the lung tissue.

Thus, as the current available screening methods for colorectal cancerare based on blood or stool samples, there is a long felt need for amethod for detecting, screening and monitoring cancers, which isapplicable for large populations without the need to use blood or stoolsamples.

It is therefore one aspect of the present invention to provide ascreening method, which is suitable to facilitate the early diagnosis ofa cancer without the need to use blood or stool samples.

It is another aspect of the present invention to provide a method formonitoring the recurrence of a cancer, status of a cancer or the effectof cancer treatment in an individual, without the need to use a blood orstool sample.

It is a third aspect of the present invention to provide a device forperforming the method according to the invention, having a simple andinexpensive design, being quick and easy to use and not requiring theassistance of specialists or the use of specialised equipment.

This is achieved according to the invention as it has surprisingly beenfound by the inventors that the TIMP-1 protein can be detected in humanexcreta, such as saliva in unexpected high concentrations with respectto the TIMP-1 concentration in plasma.

The inventors hereby overcome the common prejudice that a protein cannotbe detected in saliva if it has not been detected in urine. Aspreviously stated, earlier studies have shown, that noticeableconcentrations of TIMP-1 protein could not be detected in urine andthere has therefore been no indication in the prior art for the skilledperson to look for TIMP-1 in saliva.

Furthermore, as a non-invasive technique, the collection and analysis ofthe excreta would appear to be particularly attractive for a high-riskpatient population where the routine collecting of blood is often madedifficult because of bruised or thrombosed veins.

In addition, this would be beneficial to both the patient (bloodsampling would be reduced) and to those who handle patient samples(clinic and laboratory staff) e.g. because saliva can inhibit HIVinfectivity (Wolff and Hay, 1991).

It is therefore possible according to the present invention to providean easy and inexpensive method for early detection and monitoring of acancer in an individual or large populations using a non-invasivetechnique for collection of samples.

In order not to obtain any false-positive results it is important todetermine a discriminating value, which divides the tested individualsin a group having either a high or low likelihood of having cancer.

It has been found that in one embodiment of the invention the totalconcentration of TIMP-1 is measured, that is, the sum of the TIMP-1 infree form and the TIMP-1 in complex forms. The person skilled in the artwill understand that other expressions than the exact concentration canrepresent the concentration, such as, e.g., the concentration multipliedby a factor, etc., and that such other representations can be usedequally well for the purpose of the present invention provided thecorresponding adjustments are made.

The discriminating value is established by measuring the totalconcentration of TIMP-1 in both a healthy control population and apopulation with known cancer and thereby determining the discriminatingvalue. The discriminating value identifies the cancer population witheither a predetermined specificity or a predetermined sensitivity orboth, and is based on an analysis of the relation between theconcentration values and the known clinical data of the healthy controlpopulation and the cancer patient population.

The discriminating value determined in this manner is valid for the sameexperimental set-up in future individual tests.

Specificity is defined as the proportion of non-diseased individualshaving a parameter representing the concentration of a marker, such asTIMP-1 in excreta samples lower than a predefined level.

Sensitivity is defined as the proportion of patients having colorectalcancer having a parameter representing the concentration of a marker,e.g. TIMP-1 in excreta samples higher than a predefined level.

The method according to the invention can equally well be used formonitoring the response to treatment and the progress of the cancer asrising TIMP-1 values may suggest that a patient could have a negativedevelopment of the cancer. In such cases the discriminating value is setspecifically for each individual.

Studies have shown that an increase in detection and monitoring valuescan be obtained when combining the concentration of total TIMP-1 and theconcentration of free TIMP-1, thereby providing a highly selective andsensitive method for detecting, screening and monitoring an individualfor cancer.

Such cancers could e.g. be breast, prostate, colorectal, cervical,ovarian, lung, pancreatic, renal, vulvar, hepatocellulæar carcinomas,minimal residual disease and recurrent cancer

In order to improve the sensitivity of the method according to theinvention, an additional marker can optionally be employed. Said markeris preferably Carcino Embryonic Antigen (CEA), which is a protein, whichis normally found only during foetal development, but may reappear inadults who develop certain types of cancer, primarily cancer of thegastrointestinal system. However, other markers for colorectal cancerknown for the person skilled in the art, such as soluble u-PAR, couldequally well be employed.

Preferably logistic regression analysis can be used when combiningeither the concentration of total TIMP-1 and the concentration of freeTIMP-1 combination or the combination of concentrations of the cancermarker (CEA) and TIMP-1.

Data has shown that by adding CEA as the additional marker, animprovement in the sensitivity of total TIMP-1 in plasma can be obtainedand maintained with a very high specificity. Thus, the combination ofconcentrations of CEA and TIMP-1 could be useful in a method foridentifying patients with a high risk of having cancer.

The method according to the invention may be used both for an individualand for an entire population, but more appropriately to a populationalready identified as having an increased risk of developing cancer,e.g. individuals with a genetic disposition, individuals who have beenexposed to carcinogenic substances, or individuals withcancer-predisposing non-malignant diseases.

As an example this could e.g. be individuals with a prior polyp,individuals with Crohn's disease or ulcerative colitis, individuals withone or more family members with colorectal cancer, or individuals with aprior resection of an early colorectal cancer.

When an individual has been identified as having high TIMP-1 levels inhis or her excreta, the individual should be referred for furtherexamination. If a cancer is found, the patient could be offered surgery,radiation or adjuvant anti-neoplastic therapy aiming at curing thepatient of cancer.

The invention further relates to a dipstick for measurement of theconcentration of TIMP-1 either alone or in combination with CEA, inexcreta.

Such a dipstick is extremely easy and quick to use and each individualcan perform the test without the need of a professional staff or the useof specialist equipment.

Individuals in certain high-risk groups, such as individuals having e.g.colitis ulcerosa or individuals with an earlier detected cancer whowants to monitor a possible recurrence of the cancer, then have thepossibility of screening/monitoring themselves at any desired frequency.

Furthermore, the dipstick can be a valuable tool since each individualhas the opportunity to monitor the response to treatment and the spreadof cancer without the need for specialist equipment.

The dipstick is preferably a two-sited direct label assay in a lateralflow device format.

In a first embodiment the dipstick can be made of a capillary material,and comprises a first colour indication zone comprising antibodiesspecific for TIMP-1 and at least one reagent which can give an opticallyvisible colour change in the zone dependent on the concentration ofTIMP-1 in the tested excreta.

The dipstick further comprises a second colour indication zone, able toreact with at least one substance normally present in the excreta, andthereby providing an optically visible colour change in the zone forcontrolling that the stick is used properly.

In another embodiment of the dipstick according to the invention, thedipstick further comprises a third colour indication zone, comprisingantibodies specific for CEA and at least one reagent which can give anoptically visible colour change in the zone dependent on theconcentration of CEA in the excreta, ensuring a more reliable screeningmethod.

Preferably there is, in combinations with the dipstick, provided acolour reference that indicates the discrimination value for thespecific excreta.

When the user examines the likelihood of having a cancer, the dipstickis first brought into contact with the excreta being tested, e.g. theuser can place the dipstick in the mouth thereby bringing it intocontact with the patient's saliva.

After a certain reaction period determined by the specific reagents onthe dipstick, the user can compare the stick with the colour referencescale, dividing the patient into a group with either a high or a lowlikelihood of having cancer.

The dipstick can within the scope of the invention be based on any knownconventional detection principle based on for instance RIA or enzymaticassays.

A person skilled in the art would recognise that the dipstick is onlyone possibility of detecting TIMP-1 in excreta, such as saliva. Otheroptions within the scope of the invention are e.g. an Activity Assay(such as zymography), immunologic assays or a Colour Reaction kit.

The invention will be explained in greater detail below, by way ofexamples only with reference to the drawing, in which

FIG. 1 shows the recovery of TIMP-1 signal, measured following additionof increasing concentrations of purified TIMP-1 to a panel of 0.5% and1% saliva pools in sample dilution buffer,

FIG. 2 shows total TIMP-1 concentration from 39 dilutions of a salivapool from collection (I) assayed via ELISA, and

FIG. 3 shows the TIMP-1 concentration in each saliva sample fromcollection (I) measurement via ELISA.

EXAMPLES

It should initially be mentioned that even though the concentration ofTIMP-1 in both plasma and saliva in the following examples are presentedas ng TIMP-1 per volume plasma or saliva, it could equally well bepresented as e.g. unit or gram TIMP-1 per unit or gram of a knownreference molecule.

Donors/Patients

In collection (I), saliva was obtained from four apparently healthyvolunteer blood donors.

In collection (II) saliva was obtained form four apparently healthyvolunteer blood donors. A simultaneous plasma sample was obtained fromeach individual at the time of saliva collection.

In collection (III) saliva was obtained form three patients with knowncolon or rectal cancer. A simultaneous plasma sample was obtained fromeach individual at the time of saliva collection.

Informed consent was obtained from all donors/patients, and permissionwas obtained from the local Ethical Committees.

Saliva Collection

In collection (I) saliva was collected from healthy blood donors whowere instructed to rinse the mouth with tap water just before thecollection. The collection duration was 5 minutes and the donordelivered in this period approximately five saliva samples in a 50 mlNunc test tube. The saliva is centrifuged 5 minutes at 4000 rpm and thesupernatant is then centrifuged at 15000 rpm for 5 minutes.

In collection (II) and (III), saliva was collected by introducing acotton tampon into the mouth for 3 minutes. The tampon was thencentrifuged 5 minutes at 3000 rpm to allow the saliva to be extracted.Subsequently the saliva was centrifuged for 15.000 rpm for 5 minutes.

The resulting supernatant is in all cases frozen at −20° C., until it isused.

Blood Collection And Plasma Separation

Peripheral blood was drawn with minimal stasis (if necessary a maximumof 2 min stasis with a tourniquet at maximum +2 kPa was acceptable) intopre-chilled citrate, EDTA, or heparin collection tubes(Becton-Dickinson, Mountain View, Calif.), mixed 5 times by inversion,and immediately chilled on ice. As soon as possible (no later than 1.5 hafter collection) the plasma and blood cells were separated bycentrifugation at 4° C. at 1,200×g for 30 min, and stored frozen at −80°C. prior to assay. Plasma pools were made with freshly collected samplesfrom at least ten donors, aliquoted and stored frozen at −80° C. Foranalysis, the samples were quickly thawed in a 37° C. water bath andplaced on ice until needed.

Example 1 Measurement of Total TIMP-1 Concentration In SalivaPreparation of An ELISA To Quantitate Total TIMP-1 Concentrations InHuman Plasma

A sensitive and specific sandwich ELISA was prepared, using TIMP-1antibodies developed at the Strangeways Laboratories (Hembry et al,1985). A sheep polyclonal anti-TIMP-1 antiserum (Hembry et al, 1985;Murphy et al, 1991) was used for antigen capture, and a murinemonoclonal anti-TIMP-1 IgG1 (MAC-15) (Cooksley et al, 1990) for antigendetection.

A rabbit anti-mouse immunoglobulin/alkaline phosphatase conjugate(Catalog number D0314, Dako, Glostrup, Denmark) was the secondarydetection reagent. The latter conjugate was supplied preabsorbed againsthuman IgG, thus eliminating cross-reactivity with IgG in the samples.

As the monoclonal detection antibody MAC-15 recognises both free TIMP-1and TIMP-1 in complex with MMP's (Cooksley et al, 1990), the totalTIMP-1 content captured by the sheep polyclonal anti-TIMP-1 antiserumwas quantitated by the ELISA.

96-well microtiter plates (Maxisorp, Nunc, Roskilde, Denmark) werecoated for 1 h at 37° C. with 100 μL/well of polyclonal sheepanti-TIMP-1 (4 mg/L) in 0.1 mol/L carbonate buffer, pH 9.5. The wellswere then rinsed twice with 200 μL/well of SuperBlockJ solution (PierceChemicals, Rockford, Ill.) diluted 1:1 with phosphate-buffered saline(PBS). The microtiter plates were stored for up to 14 days at −20° C. Onthe day of analysis, the plates were thawed at room temperature andwashed 5 times in PBS containing 1 g/L Tween.

A series of purified, recombinant human TIMP-1 standards were used tocalibrate each plate. Standards were prepared by serially diluting astock solution of purified TIMP-1. Standard concentrations were 5, 3, 2,1, 0.5, 0.25, 0.1, ˜0 ng/mL. Included on each plate was a blankcontaining only sample dilution buffer, and 2 controls made from a 1:100dilution of a citrate plasma pool. One control was added as the firstsample on the plate and the second control was added as the last.

All samples were diluted 1:100 or 1:51 in sample buffer consisting of 50mol/L phosphate, pH 7.2, 0.1 mol/L NaCl, 10 g/L bovine serum albumin(Fraction V, Boehringer-Mannheim, Penzberg, Germany), and 1 g/L Tween20.

A total of 100 μL/well of each standard, blank, control, and patientsample was incubated on the plate for 1 h at 30° C. All standards,blanks, controls, and samples were run in triplicate on each plate forevery assay.

After primary incubation, the wells were washed 5 times, then treatedfor 1 h at 30° C. with 100 μL/well of purified MAC-15 monoclonalantibody (0.5 mg/L) in sample dilution buffer. After another 5 washesthe wells were incubated for 1 h at 30° C. with 100 μL/well of rabbitanti-mouse immunoglobulins(Ig)/alkaline phosphatase conjugate diluted1:2000 in sample dilution buffer.

Following 5 washes with washing solution and 3 washes with distilledwater, 100 μL of freshly made p-nitrophenyl phosphate (Sigma, St. Louis,Mo.) substrate solution (1.7 g/L in 0.1 mol/L Tris.HCl, pH 9.5, 0.1mol/L NaCl, 5 mmol/L MgCl₂) were added to each well.

The plate was placed in a Ceres 900J plate reader (Bio-Tek Instruments,Winooski, Vt.) at 23° C. with the yellow colour developmentautomatically monitored. Readings were taken at 405 nm against an airblank every 10 min. for one hour. KinetiCalc II software was used toanalyse the data by calculating the rate of colour formation for eachwell (linear regression analysis), generating a 4-parameter fittedstandard curve, and calculating the TIMP-1 concentration of each sample.

Recovery Experiments

The recovery of the TIMP-1 signal was measured following addition ofincreasing concentrations of purified TIMP-1 to a panel of 0.5% and 1%saliva pools in sample dilution buffer, followed by subsequentmeasurement of the signal.

The purified TIMP-1 was added to dilution series of the saliva pools togive concentrations in the range of 0 to 5 ng/mL.

The recovery was in each case calculated from the slope of the linerepresenting the TIMP-1 signal as a function of concentration, where100% recovery was defined as the slope obtained when TIMP-1 was dilutedin sample dilution buffer.

Recovery was 100.2% in 0.5% saliva and 100.9% in 1% saliva (FIG. 1).Thus the recovery of TIMP-1 signal from an internal standard wasexcellent for all preparations of saliva, and the saliva samples allgave good linearity of the signal as a function of dilution.

Measurement of Total TIMP-1 Concentration In Collection (I)

Saliva samples from collection (I) were pooled and 39 dilutions wereassayed for total TIMP-1 concentration via ELISA (FIG. 2). The meanTIMP-1 concentration in saliva was 72.81 ng/mL±3.33 with a referencerange from 67.29 to 79.49 ng/mL.

Each saliva sample from collection (I) as well as a pool of the sampleswas further assayed for total TIMP-1 concentration via ELISA. The meanTIMP-1 concentration in saliva was 79.66 ng/mL+3.77 with a referencerange from 76.96 to 83.96 ng/mL (FIG. 3).

Measurement of Total TIMP-1 Concentration In Collection (II)

In collection (II) both the saliva samples and the plasma samples fromhealthy blood donors were assayed for total TIMP-1 concentration viaELISA. The results are presented in table 1:

TABLE 1 Total TIMP-1 concentration (ng/mL) in saliva and plasma in fourhealthy blood donors. Total TIMP-1 Total TIMP-1 Healthy concentration inconcentration in donor saliva (ng/mL) EDTA plasma (ng/mL) 1 162 86.4 2143.8 76.5 3 87.8 82.7 4 90.5 88

Discussion

The assay described above enables accurate determination of total TIMP-1in human saliva samples. The use of a rapid blocking agent and adilution buffer with high buffering capacity also contributed toreproducible assays. Incorporating all these elements in the final assayfulfilled the requirements of sensitivity, specificity, stability, andgood recovery of an internal standard.

The quantitative studies in saliva from healthy donors showed thatsaliva samples are suitable for TIMP-1 determination.

These studies showed that for collection (I) TIMP-1, levels in saliva(mean 72-79 ng/mL) correspond to TIMP-1 levels in plasma (mean 65-70ng/mL) (see International Patent Application No. WO00/62070). Similarresults were obtained from the healthy donors in collection (II).

Example 2 Detection Value of Total TIMP-1 In Patients With ColorectalCancer

Total TIMP-1 levels in both plasma and saliva from three colorectalcancer patients (collection (III)) were measured with the TIMP-1 assaydescribed in Example 1. The TIMP-1 values were analyzed and comparedusing standard biostatistical parameters.

Patients

Three patients undergoing elective surgery for pathologically confirmedcolorectal cancer were included in the study. Blood and saliva sampleswere obtained preoperatively with informed consent from all patients inaccordance with the Helsinki declaration, and permission was granted bythe local Ethical Committee of Hvidovre Hospital, Denmark. All patientshad pathologically verified adenocarcinoma of the colon or rectum.

Measurement of Total TIMP-1 Concentration In Collection (III)

In collection (III) both the saliva samples and the plasma samples fromcolorectal patients were assayed for total TIMP-1 concentration viaELISA. The results are presented in table 2:

TABLE 2 Total TIMP-1 concentration in saliva and plasma in ng/mL inthree colorectal patients. Total TIMP-1 Total TIMP-1 Colorectalconcentration in concentration in patient saliva (ng/mL) EDTA plasma(ng/mL) 5 328 395.7 6 409.9 127 7 358.9 172.3

Discussion

This data proved that similar saliva/plasma TIMP-1 concentrations werefound not only for healthy individuals but also in patients sufferingfrom colorectal cancer.

Furthermore, all three colorectal cancer patients had elevated (abovethe 95 percentile of healthy blood donor plasma levels) saliva andplasma TIMP-1 levels. There is therefore a highly statistical differencein the total saliva TIMP-1 values between the colorectal cancer patientsand the healthy blood donors.

It has previously been found, that a highly statistical difference inthe total plasma TIMP-1 values between the colon and rectal cancerpatients each compared with the healthy blood donors exists.Furthermore, it has been shown in the aforementioned examples thatcorresponding concentrations of total TIMP-1 is present in saliva andplasma. The person skilled in the art would based on these findingtherefore expect that the same statistical difference between the colonand rectal cancer patients would be observed in total TIMP-1 fromsaliva.

On a similar basis the person skilled in the art could use the measuredtotal TIMP-1 levels in saliva from healthy donors and the colorectalcancer patients, and generate Receiver Operating Characteristics (ROC)curves to evaluate the diagnostic value of total TIMP-1. As establishedin WO00/62070, which is incorporated herein by reference, such curveshave established high sensitivity and specificity of total TIMP-1 inplasma as a marker for colorectal cancer. Based on the above findingssimilar results are expected for total TIMP-1 in saliva.

This data suggest that total TIMP-1 measurements in saliva can be usedas a screening procedure to aid in identifying patients with a high riskof having colorectal cancer.

In particular, total TIMP-1 in plasma has in WO00/62070 proven effectivein identifying patients with early cancer (Duke's stage A+B) as well asidentifying patients with more advanced disease. Based on the findingsin the present invention similar results would be expected for totalTIMP-1 concentrations in saliva. Similarly, as total TIMP-1 in plasmawas more effective in identifying patients with early stage, right-sidedcolon cancer, the same would be expected for total TIMP-1 in saliva.Right-sided colon cancer cannot be visualized by flexible sigmoidoscopy,a standard colon cancer screening methodology. It has a more insidiousonset than left-sided lesions do, and clinical symptoms develop only inthe later stages of the disease. Early diagnosis of right sided coloncancer has the potential to reduce the mortality of this disease.

The optimal discriminating value for TIMP-1 in saliva to discriminatebetween malignant and normal tissues can be determined on the basis ofsuch ROC curves as the point on the curve, which shows the optimalcombination for specificity and sensitivity and where diagnosticaccuracy is maximized. The discriminating value for total TIMP-1 inplasma based on 870 healthy blood donors has been determined (Brünner,unpublished data), and based on the above findings a similar result isexpected for total TIMP-1 in saliva.

Example 3 Measurement of the Total Concentration of TIMP-1:MMP-9Complexes And Free TIMP-1 In Saliva

As shown in Example 1 the total TIMP-1 concentration in salivacorresponds very accurately to the total TIMP-1 concentration in plasma.As it has been found that there exists a correlation between plasma andsaliva, it would be obvious to the person skilled in the art to alsoexamine the total concentration of TIMP-1:MMP-9 complexes and freeTIMP-1 concentration in saliva. Appropriate methods and materials aredescribed in similar examples in WO00/62070 said reference beingincorporated herein by reference. In view of the findings in Example 1,it would be expected that similar results would be obtained for thetotal concentration of TIMP-1:MMP-9 complexes and free TIMP-1,respectively.

Example 4 Detection Value of Total TIMP-1 In Combination With CEA InPatients With Colorectal Cancer

Examples in WO00/62070 have demonstrated that by adding an additionalmarker, an improvement in the diagnostic sensitivity of total TIMP-1 inplasma can be obtained, while maintaining a high specificity of 98%.Thus, based on the assumption that corresponding data can be obtainedwith total TIMP-1 in plasma and saliva, the combination of CEA andTIMP-1 in saliva could be useful as a screening procedure to identifypatients with a high risk of having colorectal cancer.

Example 5 Lack of Detection Value of Total TIMP-1 In Saliva In PatientsWith Primary (Stage I And II) Breast Cancer

Using the total TIMP-1 assay described in Example 1, total TIMP-1 levelsin pre-operative plasma samples from 322 patients with primary breastcancer were compared with 108 plasma samples from healthy blood donors.Data shown in WO00/62070, which is incorporated herein by reference,showed no statistical significant difference in total TIMP-1 plasmalevels between the two groups (Mann-Whitney, p=0.87). Thus, this datasupports the specificity of TIMP-1 measurements in the diagnosis ofpatients with colorectal cancer. Similar results would be expected ifthe total TIMP-1 level were measured in saliva samples from similardonors.

Example 6 Detection Value of Saliva Total TIMP-1 In Patients WithMetastatic (Stage IV) Breast Cancer

Based on information from WO00/62070 where total TIMP-1 levels weremeasured in 19 breast cancer patients with stage IV disease all EDTAplasma samples using the assay described in Example 1. These levels werecompared with total TIMP-1 plasma levels in 87 healthy female donors. AWald-Wolfowitz test indicated a highly significant difference (p<0.0001)between patient total TIMP-1 levels and those of healthy donors. As ithas been established that corresponding levels of total TIMP-1 arepresent in plasma and saliva these data show that saliva TIMP-1measurements can be used to monitor breast cancer patients forrecurrence of disease.

Example 7 Detection Value of Free TIMP-1 In Saliva In Patients WithColorectal Cancer

In WO00/62070 is was shown that free TIMP-1 in plasma alone is notlikely to be useful as a screening marker to identify patients with ahigh risk of having colorectal cancer. As previously mentioned based onthe results shown in the present application similar results areexpected for free TIMP-1 in saliva.

Example 8 Screening Value of A Dipstick For Detection of TIMP-1 InSaliva Procedure

A colloidal gold/antibody conjugate was produced for practice of themethods of the present invention. Siliconized glassware (Sigma silicote)was utilized throughout the procedure wherein 200 ml of 0.01% goldchloride (HAuCl 4.3H₂O) (Fisher Scientific, G-54-1) was brought to aboil and 2 ml of 1% sodium citrate solution was added and the boiling iscontinued for 5 minutes until the color of the solution changes frompale yellow to purple to red. A solution of potassium carbonate (0.02M)was added to the suspension in order to adjust the pH to 7.6, followedby addition of the rabbit polyclonal anti-MMP-9 antibody (1 mg/ml)(Novus Biologicals Product) such that approximately 10 μg IgG was addedper ml of gold suspension (0.01% gold).

After one minute of incubation at room temperature, 0.1 ml of a solutionof 30% bovine serum albumin in water was added to 10 ml of the goldsuspension. Aggregated material was removed by centrifugation at 3000rpm in the SS34 rotor of a Sorval RC5C centrifuge for 10 minutes. Thesupernatant was subjected to an additional centrifugation step at 6000rpm for one hour. The colloidal gold conjugate in the pellet wasresuspended in 2% bovine serum albumin in PBS (0.05 M potassiumphosphate buffer, pH 7.4, in 0.9% NaCl), the preferred conditions forliquid storage being at 4° C. Casein solution was added to give aconcentration of 1% casein immediately before application to themembrane. The conjugate was then stored for prolonged periods in the drystate, with no loss of activity after 6 months storage at 370 C. in thedry state.

According to this example, sandwich-type immunoassay devices for thedetection of TIMP-1 were constructed and used. Microporousnitrocellulose material with a thickness of approximately 0.1 mm and anaverage pore size of 5 μm was laminated with mylar and adhesive(Monokote, Top Flite Models, Inc., Chicago, Ill.). Strips measuring 1 cmby 3.5 cm were cut. Mouse monoclonal antibody MAC 19 (see WO/62070), 0.2μl, was applied to the strips at the capture zone where it wasimmobilized and air dried. Non-specific binding sites on thechromatographic strip materials were then blocked by incubation for 10minutes at room temperature with a 0.1% solution of LB gelatin in water(Inotech, Wohlen, Switzerland) and the strips were allowed to dry undera stream of air. One μl of gold particle labeled rabbit polyclonalanti-MMP-9 antibody in an anti-aggregation buffer produced as describedwas then applied to a first zone of each strip and dried.

Detection of TIMP-1 Concentration In Saliva Samples On A Dipstick

Saliva samples from collection (II) and (III), healthy blood donors andcolorectal cancer patients, respectively, were first applied to thesecond zones between the first and third zones.

The first end of the strips were then dipped into a chromatographictransport solvent comprising TBS and 1% TRITON X-100 (polyethyleneglycol tert-octylphenyl ether). The liquid front was allowed to progressto the second ends of the devices over a period of approximately 2.5minutes transporting the sample material and the gold-labeled goatanti-human IgG to the third zone.

Samples from the colorectal cancer patients (collection (III)) and theimmobilization of the labeled first reagent resulted in the presence ofa red spot at the third zone. Strips tested with samples from thehealthy blood donors, collection (II) did not produce a signal at thethird zone.

Discussion

These test confirm that TIMP-1 can be measured in saliva by means of adipstick, and that such a dipstick can be used in a screening procedureto aid in identifying patients with a high risk of having colorectalcancer. Furthermore, the dipstick can be used to monitor the response totreatment and the spread of cancer without the need for specialistequipment.

Conclusion

The person skilled in the art would understand from the presentinvention that the similarities between total TIMP-1 levels in plasmaand saliva is not limited to the Examples mentioned in the currentapplication. In fact it is now surprisingly expected that for any TIMP-1value measured in a plasma sample a corresponding value could beestablished for TIMP-1 in saliva.

This is not just true for the examples mentioned in the presentapplication or in WO00/62070 but also for all other known resultsobtained for plasma.

The data presented in the present application supports the unique valueof testing saliva when screening, monitoring and detecting early stagecolorectal cancer or metastatic breast cancer using TIMP-1 as a cancermarker. The method for identification of patients having primarycolorectal cancer is highly specific, and patients with non-malignantconditions, such as inflammatory bowl diseases, are not detected.Furthermore, saliva may also be suitable when monitoring the response totreatment and the progress of cancer.

The description has primarily described excreta from humans; however itis within the scope of the invention that the excreta is from an animal,e.g. a dog, cat or cow.

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1. A method for detecting and/or screening and/or monitoring a cancer inan individual, said method comprising determining a first parameterrepresented by the combination of the concentration of total TIMP-1 andthe concentration of free TIMP-1 in a saliva sample from the individual,wherein the presence of the first parameter above a predetermineddiscrimination value is an indication that the individual has a highlikelihood of either having a cancer or progression in a cancer.
 2. Amethod according to claim 1 wherein the cancer is selected from thegroup consisting of breast carcinoma, prostate carcinoma, colorectalcarcinoma, cervical carcinoma, ovarian carcinoma, lung carcinoma,pancreatic carcinoma, renal carcinoma, vulvar carcinoma, andhepatocellular carcinoma, minimal residual disease and recurrent cancer.3-5. (canceled)
 6. A method according to claim 1, wherein thecombination is performed by logistic regression analysis.
 7. A methodaccording to claim 1, wherein the discrimination value is determined bydetermining the total concentration of TIMP-1 in the at least oneexcreta in both a healthy control population and a population with knowncancer, thereby determining the discrimination value which identifiesthe cancer population with a predetermined specificity or apredetermined sensitivity.
 8. A method according to claim 1, wherein themethod further comprises determining at least one second parameterrepresenting the concentration of a marker for cancer different from anyform of TIMP-1, in an excreta from an individual.
 9. A method accordingto claim 8, wherein the first and second parameter are combined toresult in a combined parameter wherein the presence of a concentrationof the combined parameter above a predetermined discrimination value isan indication that the individual has a high likelihood of having acancer or that there is a progression in a cancer.
 10. A methodaccording to claim 9, wherein the predetermined discrimination value isdetermined by determining the combined parameter in the at least oneexcreta in both a healthy control population and a population with knowncolorectal cancer, thereby determining the predetermined discriminationvalue which identifies the cancer population with a predeterminedspecificity or a predetermined sensitivity.
 11. A method according toclaim 9, wherein the combination of the first and second parameter isperformed by logistic regression analysis.
 12. A method according toclaim 8, wherein the at least one second parameter is the concentrationof Carcino Embryonic Antigen (CEA).
 13. A method according to claim 7,wherein the determination of the total concentration of TIMP-1 isperformed by means of an immunoassay or an active assay.
 14. A methodaccording to claim 13, wherein the immunoassay is an ELISA.
 15. A methodaccording to claim 13, wherein the active assay is zymography.
 16. Amethod according to claim 1, which detects early stage cancer.
 17. Amethod according to claim 16, which detects early stage colorectalcancer.
 18. A method according to claim 16, which detects metastaticbreast cancer.
 19. A method according to claim 1, which monitors theresponse to cancer treatment.
 20. A method according to claim 1, whichmonitors the recurrence of a cancer.
 21. A dipstick for performing themethod according to claim 1, wherein said dipstick comprises a firstcolour indication zone, comprising antibodies specific for TIMP-1.
 22. Adipstick according to claim 21, wherein the first zone further comprisesat least one reagent which gives an optically visible colour change inthe zone dependent on the concentration of TIMP-1 in at least oneexcreta.
 23. A dipstick according to claim 21, wherein the dipstickfurther comprises a second colour indication zone, able to react with atleast one substance normally present in the excreta, and therebyproviding an optically visible colour change in the zone for controllingproper use of the dipstick.
 24. A dipstick according to claim 23,wherein said dipstick further comprises a third colour indication zone,comprising antibodies specific for CEA.
 25. A dipstick according toclaim 24, wherein the first zone further comprises at least one reagentwhich can give an optically visible colour change in the zone dependenton the concentration of CEA in at least one excreta.
 26. A methodaccording to claim 1 wherein the cancer comprises breast carcinoma,prostate carcinoma, cervical carcinoma, ovarian carcinoma, lungcarcinoma, pancreatic carcinoma, renal carcinoma, vulvar carcinoma,hepatocellulæar carcinoma, minimal residual disease or recurrent cancer.27. A method for detecting and/or screening and/or monitoring a cancerin an individual, said method comprising determining a first parameterrepresented by the combination of the concentration of total TIMP-1 andthe concentration of free TIMP-1 in a saliva sample from the individual,wherein the presence of the first parameter above a predetermineddiscrimination value is an indication that the individual has a highlikelihood of either having a cancer or progression in a cancer, andwherein the individual is selected from a population already identifiedas having an increased risk of developing cancer.
 28. A method accordingto claim 27, wherein the population includes individuals with a geneticdisposition, individuals who have been exposed to carcinogenicsubstances, or individuals with cancer-predisposing non-malignantdiseases.
 29. A method according to claim 27, wherein the individual isan individual with a prior polyp, an individual with Crohn's disease orulcerative colitis, an individual with one or more family members withcolorectal cancer, or an individual with a prior resection of an earlycolorectal cancer.
 30. A dipstick according to claim 21, which is basedon the RIA detection principle or on an enzymatic assay detectionprinciple.